Abstract Neisseria meningitidis remains a leading cause of meningitis and rapidly fatal sepsis, usually in otherwise healthy individuals. Despite effective antibiotic and supportive therapy, the mortality and morbidity of meningococcal infections remain unacceptably high. In non-epidemic periods, N. meningitidis is carried in the nasopharynx asymptomatically by 5-10% of adults, but carriage can be significantly higher (>30%) in young adult and men who have sex with men (MSM) populations. Historically, N. meningitidis has not been considered a sexually transmitted pathogen. However, several outbreaks of ST-11 serogroup C invasive meningococcal disease among MSM populations and clusters of meningococcal urethritis cases in heterosexual males have been recently documented. We provide strong microbiologic and molecular evidence indicating that one of the urethritis outbreaks, now with over 70 cases, is caused by a single capsule-defective meningococcal clonal strain belonging to the hyper-invasive ST-11 clonal complex. Seventeen percent of male urethral infections in an STI clinic initially thought to be caused by gonococci were determined to be meningococcal urethritis. Thus, potentially through oral-genital contact, ST-11 N. meningitidis now appears capable of urogenital infection like N. gonorrhoeae. The unprecedented meningococcal urethritis outbreaks suggest that this ST-11 clone has evolved novel genetic and phenotypic changes in order to effectively colonize the urogenital tract, resist local innate immune responses and cause disease, at least in males and possibly females. Importantly, we have found the meningococcal urethritis outbreak isolates demonstrated antimicrobial peptide (AMP) resistance levels that are significantly greater than gonococci and exhibited the phenomenon of ?heteroresistance? that further increased resistance to AMPs, a phenotype we report in Neisseriae for the first time. We seek, in two Specific Aims, to identify the genetic traits within the circulating meningococcal clone that are contributing to efficient male urogenital tract infection. Through detailed comparative whole genome sequencing (WGS) analyses and confirmation of pathogenic phenotypes experimentally (Aim 1), critical horizontal DNA transfer events and/or other genetic alterations will be defined. Also genotypes such as fHBP subvariants, a key antigen for the new non-capsular meningococcal vaccines, will be assessed. We will also define the antimicrobial resistance determinants that allow augmented AMP resistance and the capability to further enhance AMP resistance via the novel heteroresistance phenotype (Aim 2). Previously characterized determinants as well as those newly defined through a comprehensive random mutagenesis screen will be assessed for their contributions to AMP resistance. Potential phase variation mechanism(s) mediating heteroresistance will be assessed. By completing the Specific Aims, valuable insights into the evolution of N. meningitidis and into three NIAID key priority areas: emerging infections, sexually transmitted diseases and antimicrobial resistance, will be obtained.